Emergence of the vertebrate lineage was accompanied by the advent of the neural crest and its formation of novel derivatives, which allowed for the elaboration of the chordate body plan. While all vertebrates have neural crest cells, the neural crest is not a single population but rather comprised of distinct subpopulations, cranial, vagal, trunk and sacral, that arise at different levels of the body axis. Our preliminary results suggest that emergence of vertebrates may have occurred prior to regionalization of the body into four distinct neural crest subpopulations, since lamprey lack a discrete intermediate ?vagal? neural crest population. Thus, we hypothesize that addition of novel transcription factors via new enhancer elements in the premigratory neural crest may have conferred novel developmental potential onto this cell population, potentiating progressive expansion of the head and evolution of selected neural crest derivatives in jawed vertebrates (e.g. sympathetic, enteric neurons, jaws). To test this hypothesis and examine regulatory connections that may have facilitated production of axial level specific neural crest subpopulations, we will utilize transcriptomics, epigenomics and transgenesis approaches to perform the following aims: Specific Aim 1: Identification of lamprey homologues of ?cranial crest specific subcircuit? genes using a candidate approach. In chick, we have identified a gene regulatory subcircuit, unique to the cranial neural crest and lacking in trunk, that imbues them with the developmental potential to form craniofacial cartilage. We will test whether a homologous subcircuit exists in lamprey by isolating homologues of these genes and examining their expression pattern at premigratory, migratory and post-migratory stages. As our preliminary data suggest that several genes of this subcircuit may be ?missing? from the lamprey cranial premigratory neural crest but active later in the branchial arches, we will test the effects of ectopically introducing this subciruit at an earlier stage. Specific Aim 2: Transcriptional profiling of lamprey neural crest subpopulations along the body axis. We will perform whole population and single cell RNA-seq analysis of cranial, post-otic, trunk and sacral neural crest cells isolated by FACS from embryonic lamprey expressing crestin-enhancer mediated GFP for comparison with existing chicken and zebrafish neural crest transcriptome data sets for these axial levels. The goal is to identify transcription factors and/or genetic circuits that are absent or unique to lampreys and provide a snapshot of the transcriptional state of basal neural crest cells. Specific Aim 3: Identification of lamprey enhancers and testing their conservation via cross-species transgenesis. We will perform ATAC-seq at post-otic, trunk and sacral levels to identify open chromatin regions of lamprey neural crest cells, similar to our existing cranial ATAC dataset. For enhancer identification, we will start by testing peaks in the vicinity of 9 neural crest genes (FoxD3, Snail2, tfAP2, Ets1, Twist1, Dmbx, Lhx5, Brn3, Axud1) and expand this list as time allows. Putative enhancer regions will be tested for their ability to drive expression in the lamprey neural crest and dissected to identify their regulatory inputs. We have already identified SoxE and HoxA2 enhancers functional in the lamprey neural crest. Conserved functions will be examined via cross-species transgenesis to compare enhancer activity in lamprey and zebrafish embryos.